Efforts to develop an human immunodeficiency virus type 1 (HIV-1) vaccine that generate neutralizing antibodies have focused on antibodies that block cell-free viral transmission, yet it is increasingly clear from rcent in vitro studies that direct T cell-to-T cell transmission of HIV through a structures called virological synapses is much more efficient mode of infection. Our recent studies suggest that antibodies that can neutralize cell-free HIV-1 are often significantly less effective at neutralizing cell-associated HIV-1. Among antibodies tested, we have found that HIV-infected patient sera can be much weaker at blocking cell-associated HIV-1 when compared to homologous cell-free virus challenge. During infection through virological synapses, the viral entry receptor, Env, first functions as a cell-adhesion receptor, and secondly after a transfer into target CD4 T cells, it functions as viral membrane fusion apparatus. The conformational regulation of Env during this process may help cell-associated HIV to resist many neutralizing responses. A general hypothesis of this study is that the viral entry receptor, Env, on the surface of cells is conformationally distinct from the Env that elicits the strongest immune responses. If virological synapse-mediated infection is a predominant mode of viral dissemination in vivo, then vaccines that target more native conformations of Env on the cell-surface or virion surface, may prove more effective at blocking transmission. In this project we will employ a new method to measure antigen specific B cells that targets native conformations of Env. In this approach, fluorescent HIV-infected cells or recombinant GFP-carrying HIV-1 particles will function as antigenic probes to detect native HIV-binding B cells in patients. We will correlate the frequency of native HIV-binding memory B cells with each patient's neutralizing antibody titers against cell-to-cell or cell-free infections. Fluorescent vral clones expressing Env from different clades will measure the breadth of virus-binding responses in patients. We will further test immature virus particles that may display Env in conformations more similar to those on the surface of cells to see if these identify unique antibodies that can better neutralize cell-to-cell infection. Individual immunoglobulin (Ig) genes from HIV-specific memory B cells will be cloned from patient cells using single cell cloning approaches with coinvestigators from the Mount Sinai Center for Therapeutic Antibody Development (CTAD). We will functionally screen the resulting monoclonal antibodies for those that potently inhibit cell-to-cell mediated infection. The specific aims are as follows. Aim 1: o quantify HIV-binding memory B cells in chronically HIV-1 infected patients using fluorescent infected cells and HIV Gag-iGFP particles and to correlate this direct measure of antigen specific B cells with the ability of serum antibodies to neutralize cell-free or cell-to-cell infecion. Aim 2: To directly clone monoclonal antibodies from individual HIV-binding B cells and to examine the breadth and potency in neutralizing cell-to-cell and cell-free viral infection.